The proper growth and development of multi-cellular organisms is closely linked with cellular metabolic activity and reserve. The mismatch between metabolic supply and demand also provides many cues directing the developmental programs vital for the proper formation and function of organ systems characteristic of more complex organisms. In the central nervous system, hypoxic gradients regulate stem and neural progenitor cell survival, stimulate cell surface molecules involved in migration, and serve to fine tune the process of naturally occurring cell death through the regulated expression of neurotrophic factors (Nyakas et al., “Hypoxia and Brain Development,” Prog. Neurobiol. 49:1-51 (1996); Chen et al., “Hypoxic Microenvironment Within an Embryo Induces Apoptosis and is Essential for Proper Morphological Development,” Teratology 60:215-25 (1999); Rees et al., “Fetal and Neonatal Origins of Altered Brain Development,” Early Hum. Dev. 81:753-61 (2005); Pocock, “Oxygen Levels Affect Axon Guidance and Neuronal Migration in Caenorhabditis elegans,” Nat. Neurosci. 11:894-900 (2008)). Mild ischemia stimulates transcriptional programs that specify increased efficiency in energy utilization and activates pathways that enhance resistance to oxidant stress (Gidday, “Cerebral Preconditioning and Ischeamic Tolerance,” Nat. Rev. Neurosci. 7:437-48 (2006)). However, prolonged stress can overwhelm such innate, adaptive responses resulting in the activation of a pathological program of autophagy and delayed neuronal apoptosis (Dirnagl et al., “Pathobiology of Ischaemic Stroke: An Integrated View,” Trends Neurosci. 22:391-97 (1999)). Understanding the factors involved in regulating the balance between adaptive and pathological transcription could have far reaching implications in the fields of developmental neuroscience, cancer, and stroke therapeutics.
The basic helix loop helix transcription factor hypoxia-inducible-factor-1-α (“HIF-1α”) (GenBank Accession No. AAH12527) is central to the cellular physiological response to hypoxia and controls the expression of a broad array of genes (Semenza, “Targeting HIF-1 for Cancer Therapy,” Nat. Rev. Cancer 3:721-32 (2003)). HIF-1α plays a critical role in regulating the balance between oxidative phosphorylation and glycolysis, regulates oxygen sensing at the carotid body, and stimulates expansion of red cell mass through the transcriptional regulation of erythropoietin. In this regard, HIF-1α serves as a master vertical integrator functioning at multiple levels including the cellular, tissue, organ system, and whole organism level. Not surprising, loss of HIF-1α function results in embryonic lethality due largely to abnormalities in placental development and mesenchymal cell survival (Iyer et al., “Cellular and Developmental Control of O2 Homeostasis by Hypoxia-Inducible Factor 1 alpha,” Genes Dev. 12:149-62 (1998) and Kotch et al., “Defective Vascularization of HIF-1alpha-null Embryos is Not Associated with VEGF Deficiency but With Mesenchymal Cell Death,” Dev. Biol. 209:254-67 (1999)). In the case of ischemic stroke, HIF-1α appears to play a more complex role (Halternan et al., “Hypoxia-Inducible Factor-1alpha Mediates Hypoxia-Induced Delayed Neuronal Death that Involves p53,” J. Neurosci. 19:6818-24 (1999); Helton et al., “Brain-Specific Knock-Out of Hypoxia-Inducible Factor-1alpha Reduces Rather Than Increases Hypoxic-Ischemic Damage,” J. Neurosci. 25:4099-4107 (2005); Baranova et al., “Neuron-Specific Inactivation of the Hypoxia Inducible Factor 1 Alpha Increases Brain Injury In a Mouse Model of Transient Focal Cerebral Ischemia,” J. Neurosci. 27:6320-32 (2007)). In certain contexts, HIF-1α supports cell death signaling in neurons in concert with factors including p53 resulting in the transactivation of pro-apoptotic genes (An et al., “Stabilization of Wild-Type p53 by Hypoxia-Inducible Factor 1 Alpha,” Nature 392:405-08 (1998); Bruick, “Expression of the Gene Encoding the Proapoptic Nip3 Protein is Induced by Hypoxia,” Proc. Nat'l Acad. Sci. 97:9082-87 (2000); Kim et al., “BH3-Only Protein Noxa is a Mediator of Hypoxic Cell Death Induced by Hypoxia-Inducible Factor 1alpha,” J. Exp. Med. 199:113-24 (2004)). The molecular basis regarding HIF-1α transcriptional switching behavior remains incompletely defined.
The dual activity phosphatase MKP1/DUSP1 is regulated by hypoxia (Seta et al., “Hypoxia-Induced Regulation of MAPK Phosphatase-1 as Identified by Subtractive Suppression Hybridization and cDNA Microarray Analysis,” J. Biol. Chem. 276:44405-12 (2001)), and has been linked to the post-translational modification of HIF-1α. Like other immediate early genes including FOS and Jun, MKP-1 expression is induced early after transient forebrain ischemia in the adult (Takano et al., “Induction of CL100 Protein Tyrosine Phosphatase Following Transient Forebrain Ischemia In the Rat Brain,” J. Cereb. Blood Flow Metab. 15:33-4; Wiessner et al., “Transient Forebrain Ischemia Induces an Immediate-Early Gene Encoding the Mitogen-Activated Protein Kinase Phosphatase 3CH134 In the Adult Rat Brain,” Neuroscience 64:959-66 (1995); Nagata et al., “Profiling of Genes Associated With Transcriptional Responses In Mouse Hippocampus After Transient Forebrain Ischemia Using High-Density Oligonucleotide DNA Array,” Brain Res. Mol. Brain. Res. 121:1-11 (2004)). DUSP1 responds to oxidative stress under the transcriptional control of E2F-1 and p53 (Yang et al., “p53 Transactivates the Phosphatase MKP1 Through Both Intronic and Exonic p53 Responsive Elements,” Cancer Biol. Ther. 3:1277-82 (2004); Wu et al., “The Noncatalytic Amino Terminus of Mitogen-Activated Protein Kinase Phosphatase 1 Directs Clear Targeting and Serum Response Element Transcriptional Regulation,” Mol. Cell. Biol. 25:4792-4803 (2005); Wang et al., “Dual Specificity Phosphatase 1/CL100 is a Direct Transcriptional Target of E2F-1 in the Apoptopic Response to Oxidative Stress,” Cancer Res. 67:6737-44 (2007)). DUSP1 promotes cellular differentiation (Sakaue et al., “Role of MAPK Phosphatase-1 (MKP-1) In Adipocyte Differentiation,” J. Biol. Chem. 279:39951-57 (2004)), and reports suggest that it mediates the adaptive response of neural cells to ischemic injury (Kwak et al., “Isolation and Characterization of a Human Dual Specificity Protein-Tyrosine Phosphatase Gene,” J. Biol. Chem. 269:3596-3604 (1994)). Consistent with these findings, loss of MKP-1 activity sensitizes neuronal cells to glutamate mediated toxicity and oxidative stress in vitro (Kim et al., “MKP-1 Contributes to Oxidative Stress-Induced Apoptosis Via Inactivation of ERK1/2 In SH-SY5Y Cells,” Biochem. Biophys. Res. Commun. 338:1732-38 (2005); Choi et al., “Protein Kinase Cdelta-Mediated Proteasomal Degradation of MAP Kinase Phospohatase-1 Contributes to Glutamate-Induced Neuronal Cell Death,” J. Cell Sci. 119:1329-40 (2006)). MKP-1 is induced after BDNF-mediated stimulation of the TrkB receptor (Glorioso et al., “Specificity and Timing of Neocortical Transcriptome Changes in Response to BDNF Gene Ablation During Embryogenesis or Adulthood,” Mol. Psychiatry 11:633-48 (2006)).
The present invention overcomes limitations in the art by providing strategies aimed at modulating MKP-1's activity towards HIF, or directly regulating the machinery involved in HIF-1α cleavage, as therapeutic strategies for stroke and associated disorders in which ischemia is a central component.